Biotinylated hexadecasaccharides, preparation and use thereof

ABSTRACT

The invention concerns novel biotinylated hexadecasaccharides of general formula (I) wherein: Biot is a biotin derivative; R, R 1  and R 2 , represent independently of one another a C 1 -C 6  alkoxy or and —OSO 3 ; R 3  represents a C 1 -C 6  alkoxy or an —OSO 3 , or R 3  constitutes a —O—CH 2 — bridge; Pe represents a saccharide concatenation; as well as their pharmaceutically acceptable salts, and their use as medicines.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of co-pending applicationhaving U.S. Ser. No. 12/837,861, filed on Jul. 16, 2010, which is acontinuation of co-pending application having U.S. Ser. No. 11/684,239,filed on Mar. 9, 2007, which is continuation of International Serial No.PCT/FR05/02218, filed on Sep. 7, 2005, which claims benefit of FrenchApplication No. 0409557, filed on Sep. 9, 2004, the contents of all ofwhich are incorporated herein by reference.

The present invention relates to novel synthetic biotinylatedhexadecasaccharides having the anticoagulant and antithromboticpharmacological activities of heparin.

Heparin catalyses, especially via antithrombin III (AT III), theinhibition of two enzymes that are involved in the blood clottingcascade, namely factor Xa and factor IIa (or thrombin). Low molecularweight heparin (LMWH) preparations contain chains formed from 4 to 30monosaccharides and have the property of acting more selectively onfactor Xa than on thrombin.

It is known that the inhibition of factor Xa requires binding of heparinto AT III via the antithrombin binding domain (Domain-A) and that theinhibition of factor IIa (thrombin) requires binding to AT III, via theDomain-A, and also to thrombin via a less well defined binding domain(Domain-T).

Synthetic oligosaccharides corresponding to the Domain-A of heparin areknown. They are described, for example, in patents EP 84999 and EP 529715, the patent application published under the number WO 99/36428 andthe publication Bioorg. Med. Chem. (1998), 6, pp. 1509-1516. Thesesynthetic oligosaccharides have the property of selectively inhibiting,via AT III, the clotting factor Xa without having any activity onthrombin. They show antithrombotic activity in venous thrombosis.

Synthetic oligosaccharides capable of inhibiting thrombin and factor Xavia activation of AT III have been described in the patent applicationspublished under the numbers WO 98/03554 and WO 99/36443.

These patent applications describe novel, biologically active sulfatedand alkylated polysaccharide derivatives. They are in particularanticoagulant and antithrombotic. It has in particular been shown thatthese sulfated and alkylated polysaccharides can be powerfulantithrombotic and anticoagulant agents depending on the arrangement ofthe alkyl groups and sulfate groups borne by the carbohydrate backbone.More generally, it has been found that, by preparing polysaccharidesequences, it is possible to precisely modify the activities of GAG typeto obtain highly active products presenting the anticoagulant andantithrombotic pharmacological properties of heparin. In comparison withheparin, they have the advantage of having a determined structure and ofnot reacting with platelet factor 4, the cause of the thrombocytopaeniceffects of heparin.

However, the use in human therapeutics of some of the products describedin the patent applications published under the numbers WO 98/03554 andWO 99/36443 and in patent EP 529 715 can prove to be problematic, inparticular if these products have a long half-life. In the field of theprevention or treatment of thrombosis with the above products, thefluidity of the blood has to be reestablished or maintained while at thesame time preventing the onset of a haemorrhage.

This is because it is well known that a haemorrhage can be triggered ina patient under treatment, for any accidental reason. It may also benecessary to intervene surgically in the case of a patient underantithrombotic treatment. Furthermore, during certain surgicalprocedures, anticoagulants may be used at a high dose so as to preventthe blood from clotting, and it is necessary to neutralize them at theend of the operation. It is therefore advantageous to haveantithrombotic agents that can be neutralized in order to stop theanticoagulant activity at any time. However, the known syntheticoligosaccharides described above cannot easily be neutralized by theknown antidotes for heparin or LMWHs, including protamine sulfate.

The present invention relates to novel synthetic biotinylatedhexadecasaccharides similar in structure to the compounds described inthe patent application published under the number WO 02/24754. Thehexadecasaccharides of the invention, and also certain compoundsdescribed in document WO 02/24754, are covalently bonded to a biotinderivative (hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic acid)via a “spacer”—sequences of formula (T₁) or (T₂) as defined in thepresent patent application—and consequently have the advantage of beingable to be rapidly neutralized by a specific antidote, in an emergencysituation. This specific antidote is avidin (“The Merck Index”, Twelfthedition, 1996, M.N. 920, pp. 151-152) or streptavidin, two tetramericproteins with respective masses equal to approximately 66 000 and 60 000Da, which have very high affinity for biotin.

However, surprisingly, it appears that the length of the “spacer”linking the biotin derivative to the hexadecasaccharide chain, and alsothe position of the biotin on the saccharide unit are factors thatinfluence the efficacy of the neutralization by a specific antidote, andespecially, for example, by avidin. Thus, the hexadecasaccharidecompounds according to the invention have a much higher capacity forneutralization by a specific antidote than those described in the patentapplication published under the number WO 02/24754, by virtue of a“spacer” of controlled size and the position of the biotin on thesaccharide unit.

One subject of the present invention is biotinylated hexadecasaccharidesof general formula I:

in which:

-   -   T represents a sequence T₁ or T₂ having the following formulae:

-   -   Biot represents the group:

-   -   R represents a (C₁-C₆)alkoxy radical, especially a methoxy        radical, or an —OSO₃ ⁻ radical,    -   R₁ represents a (C₁-C₆)alkoxy radical, especially a methoxy        radical, or an —OSO₃ ⁻ radical,    -   R₂ represents a (C₁-C₆)alkoxy radical or an —OSO₃ ⁻ radical,    -   R₃ represents a (C₁-C₆)alkoxy radical, especially a methoxy        radical, or an —OSO₃ ⁻ radical, or alternatively R₃ constitutes        an —O—CH₂— bridge, the —CH₂— group being linked to the carbon        atom bearing the carboxylic function on the same ring,    -   Pe represents a saccharide sequence having the following        formula:

and the pharmaceutically acceptable salts thereof.

The polysaccharide parts consist of uncharged and/or partially chargedand/or fully charged alkylated monosaccharide units. The charged oruncharged units may be dispersed along the entire length of the chainor, in contrast, they may be grouped in charged or uncharged saccharidedomains.

In the present description, it has been chosen to represent the ¹C₄conformation for L-iduronic acid and the ⁴C₁ conformation forD-glucuronic acid, but it is well known that, in general, theconformation in solution of monosaccharide units fluctuates.

Thus, L-iduronic acid may be of ⁴C₁ ²S₀ or ⁴C₁ conformation.

The invention includes hexadecasaccharides in their acid form or in theform of any of their pharmaceutically acceptable salts. In the acidform, the —COO⁻ and —SO₃ ⁻ functions are in —COOH and —SO₃H form,respectively.

The term “pharmaceutically acceptable salt of the polysaccharides of theinvention” means a polysaccharide in which one or more of the COO⁻and/or —SO₃ ⁻ functions are ionically bonded to a pharmaceuticallyacceptable cation. The salts that are preferred according to theinvention are those whose cation is chosen from alkali metal cations andeven more preferably those whose cation is Na⁺ or K⁺.

The compounds of formula I above also comprise those in which one ormore hydrogen or carbon atoms have been replaced with the radioactiveisotope thereof, for example tritium or ¹⁴C. Such labelled compounds areuseful in research, metabolism or pharmacokinetic studies, inbiochemical tests as ligands.

In the context of the present invention, the following definitionsapply:

-   -   a (C₁-C₆)alkoxy radical: a radical —O-alkyl, the alkyl group        being a linear or branched saturated aliphatic radical        containing a chain of 1 to 6 carbon atoms. Examples of alkyl        radicals that may be mentioned include methyl, ethyl, propyl,        isopropyl, butyl, isobutyl and tert-butyl. Examples of        (C₁-C₆)alkoxy radicals that may be mentioned include methoxy and        ethoxy radicals,    -   a “spacer” T: the sequences of formula T₁ or T₂ below:

-   -   a biotinylated derivative:

The biotin derivatives are commercially available (“Pierce” catalogue1999-2000, pp. 62 to 81).

According to one of its preferred aspects, the present invention relatesto the biotinylated hexadecasaccharides of general formula I in which:

-   -   R represents a methoxy radical, or an —OSO₃ ⁻ radical,    -   R₁ represents a methoxy radical,    -   R₂ represents an —OSO₃ ⁻ radical,    -   R₃ represents a methoxy radical.

According to another of its particularly preferred aspects, theinvention relates to the following biotinylated hexadecasaccharides:

-   -   Methyl        (2,3,4,6-tetra-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-O-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-(2-[N-(6-biotinamido        hexanoyl)]-2-deoxy-3-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-β-D-glucopyranosyluronic        acid)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-L-idopyranosyluronic        acid)-(1→4)-3-O-methyl-2,6-di-O-sulfonato-α-D-glucopyranoside,        sodium salt    -   Methyl        (2,3,4,6-tetra-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)—O-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-(2-[N-(6-biotinamido        hexanoyl)]-2-deoxy-3-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-β-D-glucopyranosyluronic        acid)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-L-idopyranosyluronic        acid)-(1→4)-2,3,6-tri-O-sulfonato-α-D-glucopyranoside, sodium        salt

In its principle, the process for preparing the compounds according tothe invention uses di- or oligosaccharide base synthons prepared asreported previously in the literature. Reference will be made especiallyto the patents or patent applications EP 300 099, EP 529 715, EP 621 282and EP 649 854 and also to the documents from C. van Boeckel and M.Petitou, Angew. Chem. Int. Ed. Engl., (1993), 32, pp. 1671-1690. Thesesynthons are then coupled to one another so as to give a fully protectedequivalent of a polysaccharide according to the invention. Thisprotected equivalent is then converted into a compound according to theinvention.

One of the base synthons mentioned above contains a particular protectedfunction allowing the subsequent introduction of biotin or of a biotinderivative, for example a latent amine function in the form of an azidogroup or protected in the form of N-phthalimido.

In the coupling reactions mentioned above, a “donor” di- oroligosaccharide, activated on its anomeric carbon, reacts with an“acceptor” di- or oligosaccharide containing a free hydroxyl.

The present invention relates to a process for preparing the compoundsof formula I, characterized in that:

-   -   in a first step, a fully protected equivalent of the desired        hexadecasaccharide of formula I is obtained, containing a        protected pentasaccharide precursor, especially bearing a        suitably protected amine function for the subsequent        introduction of biotin or of a biotin derivative, this protected        pentasaccharide precursor itself being extended by a protected        precursor of the polysaccharide domain Pe;    -   in a second step, the negatively charged groups are introduced        and/or demasked;    -   in a third step, the amine function is deprotected and the        biotin or the biotin derivative is then introduced.

The pentasaccharide onto which will be grafted the biotin or the biotinderivative is synthesized according to the methods described inparticular in the patent applications published under the numbers WO98/03554 and WO 99/36443 and also in the literature (cited above).

The polysaccharide part that is the precursor of Pe is synthesizedaccording to reactions that are well known to those skilled in the art,using the methods for the synthesis of oligosaccharides (G. J. Boons,Tetrahedron, (1996), 52, pp. 1095-1121, WO 98/03554 and WO 99/36443) oran oligosaccharide when an oligosaccharide that is a glycoside bonddonor is coupled with an oligosaccharide that is a glycoside bondacceptor to give another oligosaccharide equal in size to the sum of thesizes of the two reactive species. This sequence is repeated until thecompound of formula I is obtained. The nature and profile of the chargeof the desired final compound determine the nature of the chemicalspecies used in various steps of the synthesis, according to the rulesthat are well known to those skilled in the art. Reference may be made,for example, to C. van Boeckel and M. Petitou, Angew. Chem. Int. Ed.Engl., (1993), 32, pp. 1671-1690 or to H. Paulsen, “Advances inselective chemical syntheses of complex oligosaccharides”, Angew. Chem.Int. Ed. Engl., (1982), 21, pp. 155-173.

The compounds of the invention are obtained from the fully protectedpolysaccharide precursors thereof by using the following sequence ofreactions:

-   -   the alcohol functions that are to be converted into O-sulfo        groups and the carboxylic acids are deprotected by removal of        the protecting groups used during the preparation of the        backbone, and then    -   the sulfo groups are introduced next,    -   the amine function that allows the introduction of biotin or the        biotin derivative is deprotected,    -   the biotin derivative is introduced via a standard amino/acid        coupling reaction.

The compounds of the invention may naturally be prepared by usingvarious strategies known to those skilled in the art of oligosaccharidesynthesis.

The process described above is the preferred process of the invention.However, the compounds of formula I may be prepared via other well-knownmethods of sugar chemistry described, for example, in “Monosaccharides,Their chemistry and their roles in natural products”, P. M. Collins andR. J. Ferrier, J. Wiley & Sons, (1995) and in G. J. Boons, Tetrahedron,(1996), 52, pp. 1095-1121.

The pentasaccharides Pe may thus be obtained from disaccharide synthonsas described in the publication by C. van Boeckel and M. Petitou, Angew.Chem. Int. Ed. Engl., (1993), 32, pp. 1671-1690.

The protecting groups used in the process for preparing the compounds offormula I are those commonly used in sugar chemistry, for example in“Protective Groups in Organic Synthesis”, (1981), T. W. Greene, JohnWiley & Sons, New York.

The protecting groups are advantageously chosen, for example, fromacetyl, halomethyl, benzoyl, levulinyl, benzyl, substituted benzyl,optionally substituted trityl, tetrahydropyranyl, allyl, pentenyl,tert-butyldimethylsilyl (tBDMS) and trimethylsilylethyl groups.

The activating groups are those conventionally used in sugar chemistryaccording to, for example, G. J. Boons, Tetrahedron, (1996), 52, pp.1095-1121. These activating groups are chosen, for example, fromimidates, thioglycosides, pentenylglycosides, xanthates, phosphites andhalides.

As regards both the way in which the biotin derivative is linked to theoligosaccharide and the nature of the biotin derivative, the chemicalliterature offers other possibilities that can possibly be used by meansof sets of protecting groups that are well known to those skilled in theart. Use will preferably be made of an amine function, or a thiolfunction, or a carboxylic acid function or alternatively an aldehydefunction, which will be reacted with a biotin derivative comprising areactive group of the activated ester, maleimide, iodoacetyl or primaryamine type, the reaction taking place according to the conditionsdescribed in the literature (Savage et al., “Avidin-Biotin Chemistry: AHandbook”; (1992), Pierce Chemical Company).

The process described above makes it possible to obtain the compounds ofthe invention in the form of salts. To obtain the corresponding acids,the compounds of the invention, in the form of salts, are placed incontact with a cation-exchange resin in acid form.

The compounds of the invention in acid form may then be neutralized witha base to obtain the desired salt. Any mineral or organic base thatgives pharmaceutically acceptable salts with the compounds of formula Imay be used for the preparation of the salts of the compounds of formulaI. Sodium hydroxide, potassium hydroxide, calcium hydroxide or magnesiumhydroxide is preferably used as base. The sodium and calcium salts ofthe compounds of formula I are the preferred salts.

The compounds according to the invention underwent biochemical andpharmacological studies.

1. Measurement of the Anti-factor IIa Activity and of the Anti-factor XaActivity

The circulating activity of the compounds according to the invention maybe measured by means of their anti-factor IIa activity and theanti-factor Xa activity as described by Herbert et al., Thromb Haemost.,(2001), 85(5), pp. 852-60. The compounds according to the invention areadministered intravenously (IV) or subcutaneously (SC) to rats. An IVinjection of avidin results in a large decrease in the circulatingconcentration of compound according to the invention (greater than 70%).

For example, the circulating concentration of the compound according toExample 1, after IV injection of 100 nmol/kg, is reduced by 88%(reduction measured via the anti-factor Xa activity) and 91% (reductionmeasured via the anti-factor IIa activity) 2 minutes after the IVadministration of avidin (10 mg/kg/625 nmol/kg).

The circulating concentration of the compound according to Example 2,after IV injection of 100 nmol/kg, is reduced by 76% (reduction measuredvia the anti-factor Xa activity) and 89% (reduction measured via theanti-factor IIa activity) 5 minutes after the IV administration ofavidin (10 mg/kg/625 nmol/kg).

Equivalent inhibitions for the two activities are observed when thecompounds according to Examples 1 and 2 are administered SC:

2. Measurement of the Global Antithrombotic Activity and Neutralizationwith Avidin

The global antithrombotic activity of the compounds according to theinvention and their neutralization was studied in a model of venousthrombosis consisting of an injection of tissue factor followed bystasis of rat vena cava, as described by Herbert et al., Blood, (1998),91, pp. 4197-4205.

a) Measurement of the Global Antithrombotic Activity

The compounds of the present invention are powerful thrombosisinhibitors (IC₅₀ values of less than 50 nM).

For example, Examples 1 and 2 show IC₅₀ values in this model of 3 and9.9 nM, respectively, after their IV administration.

For example, inhibition of the weight of the thrombus due to thecompound according to Example 1 (at a concentration of 30 nmol/kg) isbrought back to the control level by an IV injection of 3 mg/kg/208nmol/kg of avidin.

b) Neutralization with Avidin: Bleeding Test in Rats

The effect of compounds according to the invention was evaluated in thebleeding test in rats (Herbert et al., Blood, (1998), 91, pp.4197-4205). These compounds show high anticoagulant activity and thusincrease the bleeding time in the animal models. Avidin neutralizes theeffect of the compounds according to the invention on bleeding.

For example, the bleeding time induced in rats by 30 nmol/kg of thecompound according to Example 1 is brought back to the control level byan IV administration of avidin (3 mg/kg; 208 nmol/kg).

For example, the bleeding time induced in rats by 100 nmol/kg of thecompound according to Example 2 is brought back to the control level byan IV administration of avidin (10 mg/kg; 625 nmol/kg).

Thus, a subject of the present invention is also a process using avidinor streptavidin, characterized in that it allows the polysaccharidesaccording to the invention to be neutralized. Avidin or streptavidin maybe used for the preparation of medicaments for neutralizing thepolysaccharides according to the present invention.

By virtue of their biochemical and pharmaceutical activity, theoligosaccharides of the present invention constitute highly advantageousmedicaments. Their toxicity is entirely compatible with this use. Theyare also very stable and are thus particularly suitable for constitutingthe active principle of pharmaceutical specialties.

They can be used in various pathologies consecutive to a modification inthe homeostasis of the clotting system appearing in particular duringdisorders of the cardiovascular and cerebrovascular system, for instancethromboembolic disorders associated with atherosclerosis and diabetes,such as unstable angina, apoplexy, post-angioplasty restenosis,endarterectomy or the insertion of endovascular prostheses; orthromboembolic disorders associated with post-thrombolysis rethrombosis,infarction, dementia of ischaemic origin, peripheral arterial diseases,blood dialysis, auricular fibrillations, or alternatively during the useof vascular prostheses for aorto-coronary bypasses. These products maymoreover be used for the treatment or prevention of thromboembolicpathologies of venous origin, such as pulmonary embolisms. They may beused for preventing or treating the thrombotic complications observed,for example, following surgical operations, the growth of tumours ordisruption of clotting, induced by bacterial, viral or enzymaticactivators. In the case of their use during the insertion of prostheses,the compounds of the present invention can cover prostheses and thusmake them haemocompatible. In particular, they can be attached tointravascular prostheses (stents). In this case, they can optionally bechemically modified by introduction of a suitable arm at thenon-reducing or reducing end, as described according to EP 649 854.

The compounds of the present invention may also be used as adjuvantsduring endarterectomy performed with porous balloons.

The compounds according to the invention may be used for the preparationof medicinal products for treating the above diseases.

According to another of its aspects, a subject of the present inventionis thus a pharmaceutical composition containing, as active principle, asynthetic polysaccharide according to the invention or apharmaceutically acceptable salt thereof, optionally in combination withone or more inert and suitable excipients.

The said excipients are chosen according to the desired pharmaceuticalform and the desired mode of administration: oral, sublingual,subcutaneous, intramuscular, intravenous, transdermal, transmucous,local or rectal.

The active principle may also be presented in the form of a complex witha cyclodextrin, for example α, β or γ-cyclodextrin,2-hydroxypropyl-β-cyclodextrin or methyl-β-cyclodextrin.

The active principle may also be released by means of a ballooncontaining it or by means of an endovascular extender introduced intothe blood vessels. The pharmacological efficacy of the active principleis thus unaffected.

In each dosage unit, the active principle is present in the amountssuited to the daily doses envisaged in order to obtain the desiredprophylactic or therapeutic effect. Each dosage unit can contain from0.1 to 100 mg and preferably 0.5 to 50 mg of active principle. Thesedoses of anticoagulant compounds may be neutralized with doses of avidinor of streptavidin ranging from 1 to 1000 mg by intravenous injection,bolus or infusion.

The compounds according to the invention may also be used in combinationwith one or more other active principles that are useful for the desiredtherapy, for instance antithrombotic agents, anticoagulants, plateletaggregation inhibitors, for instance dipyridamole, aspirin, ticlopidine,clopidogrel or glycoprotein IIb/IIIa complex antagonists.

The methods, preparations and schemes below illustrate the synthesis ofthe various intermediates that are useful for obtaining thepolysaccharides according to the invention. The examples of synthesis ofhexadecasaccharides that follow illustrate the invention withoutlimiting it.

The following abbreviations are used: Bn: benzyl; Bz: benzoyl; Lev:levulinyl; Et: ethyl; Ph: phenyl; Me: methyl; Ac: acetyl; SEt:thioethyl; MP: p-methoxyphenyl; Biotin: Hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic acid; ESI: Electron Spray Ionization; TLC:thin-layer chromatography; m.p.: melting point; [α_(D)] = opticalrotation; C = concentration Rf = retention time measured on the TLCrelative to the migration solvent front.

The preparations and synthetic examples of the compounds of theinvention are detailed for illustrative purposes in the texthereinbelow.

PREPARATIONS

Preparation 1 Preparation of1,6-anhydro-2-azido-2-deoxy-4-O-p-meth-oxyphenyl-β-D-glucopyranose (No.2)

The compound 1,6:2,3-dianhydro-4-O-p-methoxy-phenyl-β-D-mannopyranose,No. 1, (4.39 g, 17.5 mmol) is synthesized by analogy with the methoddescribed in Brill and Tirefort, Tetrahedron Lett. (1998), 39, pp.787-790. Compound 1 is dissolved in 130 ml of anN,N-dimethylformamide/water mixture [4/1 (v/v)] and sodium azide (22.8g, 350 mmol) is then added. The reaction medium is heated at 120° C. for6 hours. After filtering through Celite, the filtrate is diluted withethyl acetate and then washed with water. The organic phase is driedover sodium sulfate, filtered and then concentrated under vacuum. Theresidue is recrystallized from an ethyl acetate/cyclohexane mixture (20ml/7 ml) to give 4.46 g of compound 2 in the form of crystals.

m.p.: 144° C.

Preparation 2 Preparation of1,6-anhydro-2-azido-2-deoxy-4-O-p-meth-oxyphenyl-3-O-methyl-β-D-glucopyranose(No. 3)

To a cooled (0° C.) mixture of compound 2 (4.08 g, 13.9 mmol) and methyliodide (1.1 ml, 15.3 mmol) in anhydrous N,N-dimethylformamide (40 ml) isadded portionwise sodium hydroxide (1.04 g) under an argon atmosphere.The mixture is stirred for 20 hours at room temperature. The excesssodium hydride is destroyed with methanol. After evaporating off theN,N-dimethylformamide, the residue is taken up in dichloromethane. Theorganic phase is washed with water, dried over sodium sulfate, filteredand then concentrated under vacuum. The residue is purified bychromatography on a column of silica gel [12/1 (v/v) toluene/ethylacetate] to give 3.57 g of compound 3 in the form of a white solid.

m.p.: 68° C.

Preparation 3 Preparation of1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose (No. 4)

A solution of compound 3 (8.0 g, 26.03 mmol) in 130 ml of anacetonitrile/water mixture [9/1 (v/v)] is added dropwise to a solutionof cerium ammonium nitrate (86 g, 156.2 mmol) in 390 ml of anacetonitrile/water mixture [9/1 (v/v)]. The mixture is stirred at roomtemperature for 40 minutes and then diluted with ethyl acetate. Thereaction mixture is dried over anhydrous sodium sulfate, filtered andconcentrated. Filtration on silica gel allows the cerium ammoniumnitrate residues to be partially removed. To purify the compound, anacetylation followed by a deacetylation are performed. The residue istaken up in dichloromethane (90 ml), and triethylamine (5.3 ml),dimethylaminopyridine (280 mg) and finally acetic anhydride (3.2 ml) aresuccessively added. After 16 hours, the reaction medium is diluted withdichloromethane (250 ml). The organic phase is washed with 10% potassiumhydrogen sulfate solution, with water, with 10% sodium hydrogencarbonate solution and then with water. The organic phase is then driedover anhydrous sodium sulfate, filtered and then concentrated undervacuum. The residue is purified by chromatography on a column of silicagel [10/1 (v/v) toluene/ethyl acetate] to give 4.9 g of the product insolid form. This solid is dissolved in 80 ml of adichloromethane/methanol mixture [1/1 (v/v)] and sodium methoxide (544mg) is then added. The mixture is stirred for 35 minutes and thenneutralized with a Dowex® 50WX4 H⁺ resin, filtered and concentratedunder vacuum. The residue is purified by chromatography on a column ofsilica gel [3/2 (v/v) toluene/ethyl acetate] to give 3.9 g of compound4.

[α]_(D)=−27° (C=1.06, in dichloromethane).

Preparation 4 Preparation of(2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-(1→4)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 6)

A mixture of the thioglycoside compound 5 (9.00 g, 9.04 mmol), obtainedby analogy with Preparation 1 described in the patent applicationpublished under the number WO 99/36443, compound 4 obtained inPreparation 3 (1.65 g, 8.22 mmol) and powdered 4 Å molecular sieves(9.05 g) in toluene (180 ml) is stirred under an argon atmosphere for 1hour. The mixture is then cooled to −20° C. A solution ofN-iodosuccinimide (2.14 g, 9.5 mmol) and trifluoromethanesulfonic acid(96 μl, 1.09 mmol) in 47 ml of a dichloromethane/dioxane mixture [1/1(v/v)] is added dropwise to the reaction mixture. After 10 minutes, thereaction mixture is filtered through Celite, diluted withdichloromethane (1000 ml) and washed successively with 1M sodiumthiosulfate solution, 10% sodium hydrogen carbonate solution and water.The reaction mixture is then dried over anhydrous sodium sulfate andthen concentrated under vacuum. Purification of the residue is performedby chromatography on a column of silica gel [5/1 (v/v) toluene/ethylacetate] to give 9.20 g of the trisaccharide 6.

[α]_(D)=+44° (C=1.30, in dichloromethane).

Preparation 5 Preparation of(4,6-O-benzylidene-α-D-glucopyranosyl)-(1→4)-(β-D-glucopyranosyl)-(1→4)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 7)

To a solution of compound 6 (9.2 g, 8.11 mmol) in dioxane (81 ml) isadded potassium tert-butoxide (1.82 g, 16.2 mmol). The mixture isstirred for 3 hours and then neutralized with a Dowex® 50WX4 H⁺ resin,filtered and concentrated under vacuum. After chromatography on a columnof silica gel [8/1 (v/v) dichloromethane/methanol], 5.46 g of compound 7are isolated in the form of a foam.

TLC on silica gel, dichloromethane/methanol [9/1 (v/v)]: Rf=0.35

[α]_(D)=+38° (C=0.84 in dichloromethane).

Preparation 6 Preparation of(4,6-O-benzylidene-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 8)

To a cooled (0° C.) mixture of compound 7 (5.05 g, 8.23 mmol) and methyliodide (3.8 ml, 61.7 mmol) in anhydrous N,N-dimethylformamide (150 ml)is added portionwise sodium hydroxide (1.73 g, 72.0 mmol) under an argonatmosphere. The mixture is stirred for 20 hours at room temperature. Theexcess sodium hydride is destroyed with methanol (8 ml) and the reactionmixture is poured into ice-cold water (400 ml). After extraction withethyl acetate, the organic phase is washed with saturated sodiumchloride solution, dried over anhydrous sodium sulfate and thenconcentrated under vacuum. The residue is purified by chromatography ona column of silica gel [7/1 and then 5/1 (v/v) dichloromethane/acetone]to give 4.8 g of compound 8.

[α]_(D)=+49° (C=1.02, in dichloromethane).

TLC on silica gel, dichloromethane/acetone [5/1 (v/v)]: Rf=0.45

Preparation 7 Preparation of(2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 9)

Compound 8 (5.3 g, 7.75 mmol) is dissolved in 60% acetic acid (233 ml)and stirred for 1 hour 30 minutes at 80° C. The mixture is concentratedand co-evaporated with toluene. The residue is purified bychromatography on a column of silica gel [4/1 (v/v) toluene/ethanol] togive 5.09 g of compound 9.

[α]_(D)=+57° (C=1.06 in dichloromethane).

TLC on silica gel, toluene/ethanol [4/1 (v/v)]: Rf=0.36

Preparation 8 Preparation of(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 10)

To a solution of compound 9 (5.09 g, 8.55 mmol) in dichloromethane (85ml) are added 1-benzyloxy-1H-benzotriazole (2.86 g, 11.97 mmol) andtriethylamine (1.80 ml). The mixture is stirred for 20 hours at roomtemperature and then diluted with dichloromethane. The organic phase iswashed with saturated sodium hydrogen carbonate solution and then withwater. The organic phase is then dried over anhydrous sodium sulfate,filtered and then concentrated under vacuum. The residue is purified bychromatography on a column of silica gel (15/2 (v/v) toluene/ethanol] togive 4.85 g of compound 10.

[α]_(D)=+43° (C=1.06, in dichloromethane).

TLC on silica gel, toluene/ethanol [15/2 (v/v)]: Rf=0.31

Preparation 9 Preparation of(2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-(1→4)-(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 11)

The coupling reaction of compound 10 (4.38 g, 6.26 mmol) with compound 5(“glycosyl donor deleted”), obtained by analogy with Preparation 1described in the patent application published under the number WO99/36443 (11.85 g, 11.9 mmol) is performed according to the proceduredescribed in Preparation 4, to give 8.39 g of compound 11.

[α]_(D)=+61° (C=1.06, in dichloromethane).

Preparation 10 Preparation of(4,6-O-benzylidene-α-D-glucopyranosyl)-(1→4)-(β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 12)

Compound 11 (8.36 g, 5.12 mmol), obtained in Preparation 9, is convertedinto compound 12 according to the same procedure as that described forPreparation 5. After chromatography on a column of silica gel, compound12 (4.92 g) is obtained in the form of a glass.

TLC on silica gel, dichloromethane/methanol [10/1 (v/v)]: Rf=0.41

Preparation 11 Preparation of(4,6-O-benzylidene-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 13)

Compound 12 (4.57 g, 4.54 mmol) is converted into compound 13 accordingto the same procedure as that described for Preparation 6. The crudeproduct is purified by chromatography on a column of silica gel to give4.94 g of compound 13.

[α]_(D)=+68° (C=0.93, in dichloromethane).

TLC on silica gel, toluene/ethanol [8/1 (v/v)]: Rf=0.39

Preparation 12 Preparation of(2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 14)

Compound 13 (4.89 g, 4.48 mmol) is converted into compound 14 accordingto the same procedure as that described for Preparation 7. The crudeproduct is purified by chromatography on a column of silica gel to give4.30 g of compound 14.

[α]_(D)=+80° (C=1.05, in dichloromethane).

TLC on silica gel, toluene/ethanol [4/1 (v/v)]: Rf=0.31

Preparation 13 Preparation of(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 15)

Compound 14 (4.26 g, 4.25 mmol) is converted into compound 15 accordingto the same procedure as that described for Preparation 8. The crudeproduct is purified by chromatography on a column of silica gel to give4.33 g of compound 15.

[α]_(D)=+59° (C=1.0, in dichloromethane).

TLC on silica gel, toluene/acetone [4/3 (v/v)]: Rf=0.38

Preparation 14 Preparation of(2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-(1→4)-(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 16)

The coupling reaction of the thioglycoside 5 (4.90 g, 4.93 mmol),obtained by analogy with the preparation described in the patentapplication published under the number WO 99/36443, and of compound 15(4.55 g, 4.11 mmol), described in Preparation 13, is performed accordingto the procedure described in Preparation 4. The residue obtained afterextraction is purified by chromatography on a column of silica gel togive 8.07 g of compound 16.

[α]_(D)=+71° (C=0.99, in dichloromethane).

Preparation 15 Preparation of(4,6-O-benzylidene-α-D-glucopyranosyl)-(1→4)-(β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 17)

Compound 16 is converted into compound 17 according to the proceduredescribed in Preparation 5.

TLC on silica gel, dichloromethane/methanol [8/1 (v/v)]: Rf=0.45

Preparation 16 Preparation of(4,6-O-benzylidene-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₂-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 18)

Compound 17 is converted into compound 18 according to the proceduredescribed in Preparation 6.

TLC on silica gel, toluene/acetone [5/4 (v/v)]: Rf=0.40

Preparation 17 Preparation of(2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₂-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 19)

Compound 18 is converted into compound 19 according to the proceduredescribed in Preparation 7.

TLC on silica gel, dichloromethane/methanol [10/1 (v/v)]: Rf=0.41

Preparation 18 Preparation of(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₂-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 20)

Compound 19 is converted into compound 20 according to the proceduredescribed in Preparation 8.

TLC on silica gel, cyclohexane/acetone [1/1 (v/v)]: Rf=0.36

Preparation 19 Preparation of(2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-(1→4)-(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)]₂-(1→4)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 21)

The coupling reaction of the thioglycoside 5 (3.91 g, 3.93 mmol),obtained by analogy with Preparation 1 described in the patentapplication published under the number WO 99/36443, and of compound 20(4.97 g, 3.27 mmol), obtained in Preparation 18, is performed accordingto the procedure described in Preparation 4. The crude product ispurified by chromatography on a column of silica gel to give 8.06 g ofcompound 21.

[α]_(D)=+77° (C=0.92, in dichloromethane)

Preparation 20 Preparation of(4,6-O-benzylidene-α-D-glucopyranosyl)-(1→4)-(β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)]₂-(1→4)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 22)

Compound 21 is converted into compound 22 according to the proceduredescribed in Preparation 5.

TLC on silica gel, dichloromethane/methanol [9/1 (v/v)]: Rf=0.34

Preparation 21 Preparation of(4,6-O-benzylidene-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)]₃-(1→4)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 23)

Compound 22 is converted into compound 23 according to the proceduredescribed in Preparation 6.

TLC on silica gel, cyclohexane/acetone [1/1 (v/v)]: Rf=0.44

Preparation 22 Preparation of(2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)]₃-(1→4)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 24)

Compound 23 is converted into compound 24 according to the proceduredescribed in Preparation 7.

TLC on silica gel, dichloromethane/methanol [10/1 (v/v)]: Rf=0.43

Preparation 23 Preparation of(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)]₃-(1→4)-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 25)

Compound 24 is converted into compound 25 according to the proceduredescribed in Preparation 8.

TLC on silica gel, dichloromethane/methanol [10/1 (v/v)]: Rf=0.64

Preparation 24 Preparation of(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-1,6-anhydro-2-azido-2-deoxy-3-O-methyl-β-D-glucopyranose(No. 27)

A mixture of the thioglycoside compound 26 (10.1 g, 10.4 mmol), preparedby analogy with the method described in Preparation 36 of the patentapplication published under the number WO 99/36443, of the acceptorcompound 25 (5.02 g, 2.61 mmol) obtained in Preparation 23 and ofpowdered 4 Å molecular sieves (14.5 g) in toluene (220 ml) is stirredunder an argon atmosphere for 1 hour. The reaction mixture is cooled to0° C. and a solution of N-iodosuccinimide (2.58 g) andtrifluoromethanesulfonic acid (366 μl) in 57 ml of adichloromethane/dioxane mixture [1/1 (v/v)] is introduced therein. After40 minutes, the mixture is filtered through Celite, diluted with tolueneand successively washed with 1M sodium thiosulfate solution, 10% sodiumhydrogen carbonate solution and water. The reaction mixture is thendried over anhydrous sodium sulfate, filtered and then concentratedunder vacuum. The residue is purified by chromatography on a column ofsilica gel [17/1/1 and then 14/1/1 (v/v/v) dichloromethane/ethylacetate/ethanol] to give 5.87 g of compound 27.

TLC on silica gel, dichloromethane/ethyl acetate/ethanol [8/0.5/0.5(v/v/v)]; [toluene/acetone (1/1 (v/v)]: Rf=0.41

Preparation 25 Preparation of(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)(1→4)-(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-1,6-di-O-acetyl-2-azido-2-deoxy-3-O-methyl-D-glucopyranose(No. 28)

A solution of compound 27 (4.06 g, 1.44 mmol) in a mixture of aceticanhydride (13.6 ml) and trifluoroacetic acid (1.2 ml) is stirred for 6hours. After concentrating, the mixture is co-evaporated with toluene(5×25 ml). The residue is purified by chromatography on a column ofsilica gel [3/1/1 (v/v/v) cyclohexane/ethyl acetate/ethanol] to give2.930 g of compound 28.

TLC on silica gel, toluene/acetone [1/1 (v/v)]: Rf=0.44

Preparation 26 Preparation of(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-6-O-acetyl-2-azido-2-deoxy-3-O-methyl-D-glucopyranose(No. 29)

A solution of compound 28 (2.72 g, 0.928 mmol) and of benzylamine (3.9ml, 35.2 mmol) in tetrahydrofuran is stirred at room temperature for 16hours. The reaction mixture is diluted with ethyl acetate, washed with1M hydrochloric acid and with water, dried over sodium sulfate, filteredand then concentrated under vacuum. Purification of the residue bychromatography on a column of silica gel [4/5 (v/v) cyclohexane/acetone]gives 2.21 g of compound 29.

TLC on silica gel, cyclohexane/acetone [4/5 (v/v)]: Rf=0.42

Preparation 27 Preparation of(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1-+4)-(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1-+4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1-4.4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-6-O-acetyl-2-azido-2-deoxy-3-O-methyl-D-glucopyranosetrichloroacetimidate (No. 30)

Trichloroacetonitrile (54.6 μl, 541 μmol) and caesium carbonate (56.4mg, 173 μmol) are added to a solution of compound 29 (0.313 g, 108 μmol)in dichloromethane (3 ml). After stirring for 1 hour 30 minutes, themixture is filtered and then concentrated. The residue is purified bychromatography on a column of silica gel, using for the elution amixture of cyclohexane/ethyl acetate/ethanol [2/0.5/0.5 (v/v/v)]containing 0.1% triethylamine, to give 245 mg of compound 30.

TLC on silica gel, cyclohexane/ethyl acetate/ethanol [5/1.5/1.5(v/v/v)]: Rf=0.41

Preparation 28 Preparation of methyl(4-O-levulinyl-2,3-di-O-methyl-β-D-glucopyranosyluronicacid)-(1→4)-(3,6-di-O-acetyl-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-L-idopyranosyluronicacid)-(1→4)-3-O-methyl-α-D-glucopyranoside (No. 32)

A solution of compound 31 (4.50 g, 3.02 mmol), prepared by analogy withPreparation 31 described in the patent application published under thenumber WO 99/36443, in 72 ml of an ethyl acetate/tert-butanol mixture[1/1 (v/v)] is treated under a pressure of hydrogen (4 bar) in thepresence of 10% palladium-on-charcoal (9.0 g) for 6 hours. Afterfiltration and concentration, the compound 32 obtained is used directlyin the following step without purification.

Preparation 29 Preparation of methyl (methyl4-O-levulinyl-2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1→4)-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1→4)-(methyl2,3-di-O-methyl-α-L-idopyranosyluronate)-(1→4)-2,6-di-O-acetyl-3-O-methyl-α-D-glucopyranoside(No. 33)

To a solution of compound 32 (1.09 g, 1.13 mmol) in anhydrousN,N-dimethylformamide (15 ml) are added, at 0° C., potassium hydrogencarbonate (1.13 g) and then methyl iodide (1.4 ml). After stirring for16 hours at room temperature, the reaction medium is cooled to 0° C.Dimethylaminopyridine (44 mg) and then acetic anhydride (2.4 ml) arethen successively added. The mixture is stirred for 16 hours. Afterneutralizing the excess acetic anhydride, the mixture is diluted withethyl acetate. The organic phase is successively washed with 10%potassium hydrogen sulfate solution, with water and then with saturatedsodium hydrogen carbonate solution and with water. The organic phase isthen dried over anhydrous sodium sulfate, filtered and then evaporatedto dryness. The residue obtained is subjected to acetylation again underthe standard conditions (acetic anhydride, dimethylaminopyridine,triethylamine in dichloromethane). After work-up, the residue ispurified by chromatography on a column of silica gel [12/2.5/2.5 (v/v/v)cyclohexane/ethyl acetate/ethanol] to give 0.910 g of compound 33.

TLC on silica gel, toluene/acetone [2/1 (v/v)]: Rf=0.49

Preparation 30 Preparation of methyl (methyl2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1→4)-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1→4)-(methyl2,3-di-O-methyl-α-L-idopyranosyluronate)-(1→4)-2,6-di-O-acetyl-3-O-methyl-α-D-glucopyranoside(No. 34)

Compound 33 (0.884 g, 0.793 mmol) is dissolved in 160 ml of atoluene/ethanol mixture [1/1 (v/v)]. Hydrazine acetate (0.365 mg) isadded. After stirring for 5 hours at room temperature, the reactionmedium is concentrated to dryness. The residue is dissolved indichloromethane. The organic phase is successively washed with 2% sodiumhydrogen carbonate solution and with water, and then dried overanhydrous sodium sulfate, filtered and evaporated to dryness. Afterchromatography on a column of silica gel [5/3 (v/v) toluene/acetone],0.696 g of compound 34 is obtained.

TLC on silica gel, toluene/acetone [2/1 (v/v)]: Rf=0.37

Preparation 31 Preparation of methyl(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)—O-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-(6-O-acetyl-2-azido-2-deoxy-3-O-methyl-α-D-glucopyranosyl)-(1→4)-(methyl2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1→4)-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1→4)-(methyl2,3-di-O-methyl-α-L-idopyranosyluronate)-(1→4)-2,6-di-O-acetyl-3-O-methyl-α-D-glucopyranoside(No. 36)

The imidate compound 30 (170 mg, 56 μmol) obtained in Preparation 27,and compound 34 (114 mg, 112 μmol) obtained in Preparation 30 aredissolved in 2.5 ml of a dichloromethane/diethyl ether mixture [1/2(v/v)]. After addition of powdered 4 Å molecular sieves, the mixture iscooled to −20° C. and a 0.1M solution of trimethylsilyltrifluoromethanesulfonate in dichloromethane (84 μl) is added. After 40minutes, the mixture is neutralized by addition of solid sodium hydrogencarbonate. After filtration and concentration, the residue is purifiedby chromatography on Sephadex® LH60 gel, followed by chromatography on acolumn of silica gel [diethyl ether/ethanol (17/2 v/v)] to give 123 mgof compound 36.

Mass: “ESI” method, positive mode: chemical mass=3890.87; experimentalmass: 3890.46±0.68 a.m.u.

TLC on silica gel, diethyl ether/ethanol [17/2 (v/v)]: Rf=0.40

Preparation 32 Preparation of methyl(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-(1→4)-(6-O-benzoyl-2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)—O-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-(6-O-acetyl-2-azido-2-deoxy-3-O-methyl-α-D-glucopyranosyl)-(1→4)-(methyl2,3-di-O-methyl-β-D-glucopyranosyluronate)-(1→4)-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1→4)-(methyl2,3-di-O-methyl-α-L-idopyranosyluronate)-(1→4)-2,3,6-tri-O-acetyl-α-D-glucopyranoside(No. 37)

The imidate compound 30 obtained according to Preparation 27 (95 mg,0.031 mmol) and compound 35 (65.4 mg, 0.062 mmol) obtained inPreparation 42 of the patent application published under the number WO02/24754 are dissolved in 1.5 ml of a dichloromethane/diethyl ethermixture [1/2 (v/v)]. After addition of powdered 4 Å molecular sieves,the mixture is cooled to −20° C. and a 0.1M solution of trimethylsilyltrifluoromethanesulfonate in dichloromethane (47 μl) is added. After 40minutes, the mixture is neutralized by addition of solid sodium hydrogencarbonate. After filtration and concentration, the residue is purifiedby chromatography on Sephadex LH60 gel (2 chromatographies wereperformed) to give 68 mg of compound 37.

Mass: “ESI” method, positive mode: chemical mass=3918.88; experimentalmass: 3919.35±0.71 a.m.u.

TLC on silica gel, cyclohexane/ethyl acetate/ethanol [3/1/1 (v/v/v)]:Rf=0.53

Preparation 33 Preparation of methyl(α-D-glucopyranosyl)-(1→4)-(α-D-glucopyranosyl)-(1→4)-(β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)—O-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-(2-azido-2-deoxy-3-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-β-D-glucopyranosyluronicacid)-(1→4)-(α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-L-idopyranosyluronicacid)-(1→4)-3-O-methyl-α-D-glucopyranoside (No. 38)

30% aqueous hydrogen peroxide solution (3.9 ml) is added, at −5° C., toa solution of compound 36 (111 mg) obtained in Preparation 31 intetrahydrofuran (4.6 ml). After stirring for 5 minutes, aqueous 0.7Mlithium hydroxide solution (1.8 ml) is added dropwise. The reactionmixture is stirred for 1 hour at −5° C. and then for 4 hours at 0° C.and finally for 16 hours at room temperature. The reaction mixture isdeposited on a column of fine Sephadex® G-25 (5×100 cm) eluted withwater. The fractions containing the expected compound are combined,concentrated and deposited on a column of Dowex® AG 50 WX4 H⁺ resin (1.9ml). The compound is collected at 0° C. and concentrated to give 72.1 mgof compound 38.

TLC on silica gel, ethyl acetate/pyridine/acetic acid/water[16/12/2.6/7(v/v/v/v)]: Rf=0.60.

Preparation 34 Preparation of methyl(α-D-glucopyranosyl)-(1→4)-(α-D-glucopyranosyl)-(1→4)-(β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)—O-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-(2-azido-2-deoxy-3-O-methyl-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-β-D-glucopyranosyluronicacid)-(1→4)-(α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-L-idopyranosyluronicacid)-(1→4)-α-D-glucopyranoside (No. 39)

30% aqueous hydrogen peroxide solution (2.2 ml) is added, at −5° C., toa solution of compound 37 (60 mg) in tetrahydrofuran (5.5 ml). Afterstirring for 5 minutes, aqueous 0.7M lithium hydroxide solution (1 ml)is added dropwise. The reaction mixture is stirred for 1 hour at −5° C.,then for 4 hours at 0° C. and finally for 16 hours at room temperature.The mixture is neutralized with 1M hydrochloric acid solution. Thesolution is deposited on a column of fine Sephadex® G-25 (5×100 cm)eluted with water. The fractions containing the expected compound arecombined, concentrated and deposited on a column of Dowex® AG 50 WX4 H⁺resin. The compound is collected at 0° C. and concentrated to give 37.5mg of compound 39.

TLC on silica gel, ethyl acetate/pyridine/acetic acid/water[16/12/2.6/7(v/v/v/v)]: Rf=0.36.

Preparation 35 Preparation of methyl(2,3,4,6-tetra-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)—O-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-(2-azido-2-deoxy-3-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-β-D-glucopyranosyluronicacid)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-L-idopyranosyluronicacid)-(1→4)-3-O-methyl-2,6-di-O-sulfonato-α-D-glucopyranoside, sodiumsalt (No. 40)

Just before using it, compound 38 obtained in Preparation 33 isco-distilled with N,N-dimethylformamide (3×2 ml). To a solution ofcompound 38 (70.3 mg, 22.8 μmol) in N,N-dimethylformamide (2 ml) isadded sulfur trioxide-triethylamine complex (351 mg). The mixture isstirred for 16 hours at 55° C. in the absence of light. The mixture,cooled to 0° C., is added dropwise to a solution of sodium hydrogencarbonate in water. The resulting mixture is stirred for 16 hours atroom temperature and concentrated to dryness. The residue, dissolved inwater, is deposited on a column of fine Sephadex G-25 eluted with 0.2Msodium chloride. The fractions containing the product are concentratedand desalified using the same column eluted with water. Afterfreeze-drying, 103 mg of compound 40 are obtained.

Mass: “ESI” method, negative mode: chemical mass=4864.82; experimentalmass: 4862.90±0.21 a.m.u.

Preparation 36 Preparation of methyl(2,3,4,6-tetra-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-6-O-sulfonato-(α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)—O-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-(2-azido-2-deoxy-3-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-β-D-glucopyranosyluronicacid)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-L-idopyranosyluronicacid)-(1→4)-2,3,6-tri-O-sulfonato-α-D-glucopyranoside, sodium salt (No.41)

Compound 39 (33 mg, 0.017 mmol) obtained in Preparation 34 is convertedinto compound 41 according to the procedure described in Preparation 35.After concentration, 40 mg of compound 41 are obtained.

Mass: “ESI” method, negative mode: chemical mass=4952.83; experimentalmass: 4950.19±0.55 a.m.u.

Preparation 37 Preparation of methyl(2,3,4,6-tetra-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)—O-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-(2-amino-2-deoxy-3-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-β-D-glucopyranosyluronicacid)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-L-idopyranosyluronicacid)-(1→4)-3-O-methyl-2,6-di-O-sulfonato-α-D-glucopyranoside, sodiumsalt (No. 42)

A solution of compound 40 (93.8 mg) obtained in Preparation 35 in amixture of tert-butanol (1.2 ml) and water (1.8 ml) is treated under apressure of hydrogen (5 bar) in the presence of 10%palladium-on-charcoal (28 mg) at 40° C. for 4 hours. After filtration(Millipore® LSWP 5 μm filter), the solution is concentrated to drynessto give 93 mg of compound 42.

Preparation 38 Preparation of methyl(2,3,4,6-tetra-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)—O-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-(2-amino-2-deoxy-3-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-β-D-glucopyranosyluronicacid)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-L-idopyranosyluronicacid)-(1→4)-2,3,6-tri-O-sulfonato-α-D-glucopyranoside, sodium salt (No.43)

Compound 41 (40.8 mg) obtained in Preparation 36 is converted intocompound 43 according to the procedure described in Preparation 37.After concentration, 42.3 mg of compound 43 are obtained.

Mass: “ESI” method, negative mode: chemical mass=4926.84; experimentalmass: 4924.07±0.36 a.m.u.

Example 1

Example 2

Example 1 Preparation of methyl(2,3,4,6-tetra-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)—O-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-(2-[N-(6-biotinamidohexanoyl)]-2-deoxy-3-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-β-D-glucopyranosyluronicacid)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-L-idopyranosyluronicacid)-(1→4)-3-O-methyl-2,6-di-O-sulfonato-α-D-glucopyranoside, sodiumsalt

Compound 42 (20 mg, 4.13 μmol) obtained in Preparation 37 is dissolvedin aqueous 0.5% sodium hydrogen carbonate solution (1.7 ml). A solutionof sulfosuccinimide 6-(biotinamido)hexanoate (23 mg, 41.3 μmol) in 0.5%sodium hydrogen carbonate solution (100 μL) is added dropwise thereto.After stirring for 16 hours at room temperature, aqueous 1M sodiumhydroxide solution is added and the mixture is stirred for 1 hour. Thereaction mixture is deposited on a column of fine Sephadex® G-25 (5×100cm) eluted with aqueous 0.2M sodium chloride solution. The fractionscontaining the product are concentrated and desalified using the samecolumn eluted with water. After freeze-drying, 21.2 mg of the compoundof Example 1 are obtained.

Mass: “ESI” method, negative mode; monoisotopic mass=5174.38; chemicalmass=5178.28; experimental mass=5177.69±0.52 a.m.u.

Example 2 Preparation of methyl(2,3,4,6-tetra-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-sulfonato-β-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)-[(2,3,6-tri-O-methyl-α-D-glucopyranosyl)-(1→4)—O-(2,3,6-tri-O-methyl-β-D-glucopyranosyl)-(1→4)]₃-(2-[N-(6-biotinamidohexanoyl)]-2-deoxy-3-O-methyl-6-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-β-D-glucopyranosyluronicacid)-(1→4)-(2,3,6-tri-O-sulfonato-α-D-glucopyranosyl)-(1→4)-(2,3-di-O-methyl-α-L-idopyranosyluronicacid)-(1→4)-2,3,6-tri-O-sulfonato-α-D-glucopyranoside, sodium salt

Compound 43 (19.1 mg) obtained according to Preparation 38 is convertedinto compound 45 according to the procedure described in Example 1.After freeze-drying, 18.1 mg of the compound of Example 2 are obtained.

Mass: “ESI” method, negative mode: monoisotopic mass=5262.31; chemicalmass=5266.30; experimental mass: 5263.93±0.38 a.m.u.

What is claimed is:
 1. A method of treating a pathology consecutive to amodification in the homeostasis of the clotting system appearing duringdisorders of the cardiovascular and cerebrovascular system, comprising:administering to a patient in need thereof an effective amount of abiotinylated hexadecasaccharide of general formula I:

in which: T represents a sequence T₁ or T₂ having the followingformulae:

Biot represents the group:

R represents a (C₁-C₆)alkoxy radical, or an —OSO₃ ⁻ radical; R₁represents a (C₁-C₆)alkoxy radical, or an —OSO₃ ⁻ radical; R₂ representsa (C₁-C₆)alkoxy radical or an —OSO₃ ⁻ radical; R₃ represents a(C₁-C₆)alkoxy radical, or an —OSO₃ ⁻ radical, or alternatively R₃constitutes an —O—CH₂— bridge, the —CH₂— group being linked to thecarbon atom bearing the carboxylic function on the same ring; and Perepresents a saccharide sequence having the following formula:

and a pharmaceutically acceptable salt thereof; wherein the pathology isselected from: unstable angina, apoplexy, post-angioplasty restenosis,thromboembolic disorders associated with post-thrombolysis rethrombosis,infarction, dementia of ischaemic origin, peripheral arterial diseases,blood dialysis, auricular fibrillations, thrombosis during the use ofvascular prostheses for aorto-coronary bypasses, thromboembolicpathologies of venous origin, pulmonary embolism, thromboticcomplications observed following surgical operations, the development oftumours or the disruption of clotting, induced by bacterial, viral orenzymatic activators and thrombosis associated with endarterectomy orthe insertion of endovascular prostheses.
 2. A method of treating aprosthesis, comprising: covering the prosthesis with a biotinylatedhexadecasaccharide according to claim
 1. 3. A method of performing anendarterectomy performed with a porous balloon, comprising: using abiotinylated hexadecasaccharide according to claim 1 as an adjuvant.